The deubiquitinating enzyme USP19 facilitates hepatocellular carcinoma progression through stabilizing YAP.

Hippo pathway plays a crucial role in the progression of hepatocellular carcinoma (HCC), and yes-associated protein (YAP) is one of the major factors of the Hippo pathway. However, the mechanism of abnormal YAP activation in HCC has not been well elucidated. Here, we screened a Deubiquitinating enzymes' (DUB) siRNA library targeting DUBs, and identified Ubiquitin Specific Peptidase 19 (USP19) as a specific deubiquitinating enzyme of YAP in HCC, which could stabilize YAP at K76 and K90 sites via removing the K48- and K11-linked ubiquitin chains. USP19 knockdown decreased the expression of YAP protein and its target gene (CTGF, CYR61, ANKRD1) expression. Through substantial in vivo and in vitro experiments, we prove that USP19 facilities the proliferation and migration of HCC. More importantly, we found that USP19 was upregulated in HCC tissues and associated with poor prognosis. In general, our research revealed a novel post-translational mechanism between USP19 and YAP in HCC, suggesting that USP19 may be a pivotal therapeutic target for HCC treatment.

Short-term tamoxifen administration improves hepatic steatosis and glucose intolerance through JNK/MAPK in mice.

Nonalcoholic fatty liver disease (NAFLD) which is a leading cause of chronic liver diseases lacks effective treatment. Tamoxifen has been proven to be the first-line chemotherapy for several solid tumors in clinics, however, its therapeutic role in NAFLD has never been elucidated before. In vitro experiments, tamoxifen protected hepatocytes against sodium palmitate-induced lipotoxicity. In male and female mice fed with normal diets, continuous tamoxifen administration inhibited lipid accumulation in liver, and improved glucose and insulin intolerance. Short-term tamoxifen administration largely improved hepatic steatosis and insulin resistance, however, the phenotypes manifesting inflammation and fibrosis remained unchanged in abovementioned models. In addition, mRNA expressions of genes related to lipogenesis, inflammation, and fibrosis were downregulated by tamoxifen treatment. Moreover, the therapeutic effect of tamoxifen on NAFLD was not gender or ER dependent, as male and female mice with metabolic disorders shared no difference in response to tamoxifen and ER antagonist (fulvestrant) did not abolish its therapeutic effect as well. Mechanistically, RNA sequence of hepatocytes isolated from fatty liver revealed that JNK/MAPK signaling pathway was inactivated by tamoxifen. Pharmacological JNK activator (anisomycin) partially deprived the therapeutic role of tamoxifen in treating hepatic steatosis, proving tamoxifen improved NAFLD in a JNK/MAPK signaling-dependent manner.

Molecular pathogenesis: Connections between viral hepatitis-induced and non-alcoholic steatohepatitis-induced hepatocellular carcinoma.

Hepatocellular carcinoma(HCC) is the sixth most common cancer in the world and is usually caused by viral hepatitis (HBV and HCV), alcoholic, and non-alcoholic fatty liver disease(NAFLD). Viral hepatitis accounts for 80% of HCC cases worldwide. In addition, With the increasing incidence of metabolic diseases, NAFLD is now the most common liver disease and a major risk factor for HCC in most developed countries. This review mainly described the specificity and similarity between the pathogenesis of viral hepatitis(HBV and HCV)-induced HCC and NAFLD-induced HCC. In general, viral hepatitis promotes HCC development mainly through specific encoded viral proteins. HBV can also exert its tumor-promoting mechanism by integrating into the host chromosome, while HCV cannot. Viral hepatitis-related HCC and NASH-related HCC differ in terms of genetic factors, and epigenetic modifications (DNA methylation, histone modifications, and microRNA effects). In addition, both of them can lead to HCC progression through abnormal lipid metabolism, persistent inflammatory response, immune and intestinal microbiome dysregulation.

Prognostic value of preoperative inflammatory markers in patients with hepatocellular carcinoma who underwent curative resection.

BACKGROUND: The prognosis of hepatocellular carcinoma (HCC) is not optimistic. Our study focused on present inflammatory markers, including the neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), gamma-glutamyl transpeptidase-to-platelet ratio (GPR), aspartate aminotransferase-to-lymphocyte ratio (ALR) and fibrinogen-to-albumin ratio (FAR), and explored their optimal combination for the prognosis of HCC after resection. METHODS: A total of 347 HCC patients who underwent curative resection were enrolled. The optimal cutoff values of the inflammatory markers were calculated using receiver operating characteristic (ROC) curve analysis, and used to divide patients into two groups whose differences were compared by Kaplan-Meier analysis. Cox univariate and multivariate analyses were used to analyze the independent prognostic inflammatory markers. The χ2 test was chosen to determine the relationship between independent prognostic inflammatory markers and clinicopathological features. We created combined scoring models and evaluated them by Cox univariate and multivariate methods. The concordance index (C-index), Akaike information criterion (AIC) and likelihood ratio were calculated to compare the models. The selected optimal inflammatory markers and their combinations were tested in different stages of HCC by Kaplan-Meier analysis. RESULTS: The ALR and GPR were independent prognostic factors for disease-free survival (DFS); the ALR, PLR, and GPR were independent prognostic factors for overall survival (OS). The proposed GPR and ALR-GPR-PLR score models were independent predictors for DFS and OS, respectively. CONCLUSION: The preoperative GPR and ALR-GPR-PLR score models were independent predictors for DFS and OS, respectively, and performed well in stratifying patients with HCC. The higher the score in the model was, the worse the prognosis.

GDF15 induces immunosuppression via CD48 on regulatory T cells in hepatocellular carcinoma.

BACKGROUND: A better understanding of the molecular mechanisms that manifest in the immunosuppressive tumor microenvironment (TME) is crucial for developing more efficacious immunotherapies for hepatocellular carcinoma (HCC), which has a poor response to current immunotherapies. Regulatory T (Treg) cells are key mediators of HCC-associated immunosuppression. We investigated the selective mechanism exploited by HCC that lead to Treg cells expansion and to find more efficacious immunotherapies. METHODS: We used matched tumor tissues and blood samples from 150 patients with HCC to identify key factors of Treg cells expansion. We used mass cytometry (CyTOF) and orthotopic cancer mouse models to analyze overall immunological changes after growth differentiation factor 15 (GDF15) gene ablation in HCC. We used flow cytometry, coimmunoprecipitation, RNA sequencing, mass spectrum, chromatin immunoprecipitation and Gdf15-/-, OT-I and GFP transgenic mice to demonstrate the effects of GDF15 on Treg cells and related molecular mechanism. We used hybridoma technology to generate monoclonal antibody to block GDF15 and evaluate its effects on HCC-associated immunosuppression. RESULTS: GDF15 is positively associated with the elevation of Treg cell frequencies in patients wih HCC. Gene ablation of GDF15 in HCC can convert an immunosuppressive TME to an inflammatory state. GDF15 promotes the generation of peripherally derived inducible Treg (iTreg) cells and enhances the suppressive function of natural Treg (nTreg) cells by interacting with a previously unrecognized receptor CD48 on T cells and thus downregulates STUB1, an E3 ligase that mediates forkhead box P3 (FOXP3) protein degradation. GDF15 neutralizing antibody effectively eradicates HCC and augments the antitumor immunity in mouse. CONCLUSIONS: Our results reveal the generation and function enhancement of Treg cells induced by GDF15 is a new mechanism for HCC-related immunosuppression. CD48 is the first discovered receptor of GDF15 in the immune system which provide the possibility to solve the molecular mechanism of the immunomodulatory function of GDF15. The therapeutic GDF15 blockade achieves HCC clearance without obvious adverse events.

Tripartite motif 16 ameliorates nonalcoholic steatohepatitis by promoting the degradation of phospho-TAK1.

Nonalcoholic steatohepatitis (NASH)-related hepatocellular carcinoma and liver disorders have become the leading causes for the need of liver transplantation in developed countries. Lipotoxicity plays a central role in NASH progression by causing endoplasmic reticulum stress and disrupting protein homeostasis. To identify key molecules that mitigate the detrimental consequences of lipotoxicity, we performed integrative multiomics analysis and identified the E3 ligase tripartite motif 16 (TRIM16) as a candidate molecule. In particular, we found that lipid accumulation and inflammation in a mouse NASH model is mitigated by TRIM16 overexpression but aggravated by its depletion. Multiomics analysis showed that TRIM16 suppressed NASH progression by attenuating the activation of the mitogen-activated protein kinase (MAPK) signaling pathway; specifically, by preferentially interacting with phospho-TAK1 to promote its degradation. Together, these results identify TRIM16 as a promising therapeutic target for the treatment of NASH.

The mRNA of TCTP functions as a sponge to maintain homeostasis of TCTP protein levels in hepatocellular carcinoma.

Translationally controlled tumor protein (TCTP) is a highly conserved protein that accumulated in the tumorigenesis of various malignancies. Despite the important role of TCTP protein in tumor progression, the precise function and underlying mechanistic regulation of TCTP mRNA in hepatocellular carcinoma (HCC) remain unclear. In this study, we found that TCTP protein was overexpressed in HCC patients but TCTP mRNA expression levels were reversed. TCTP knockout HCC cells exhibited attenuated abilities of proliferation, migration, and invasion. The knockdown of TCTP by siRNA effectively reduced TCTP mRNA levels but not protein levels in HCC cells. Moreover, although the constitutive knockdown of TCTP inhibited almost 80% of TCTP protein expression levels in tumors of wildtype transgenic mice (TCTP KD/WT), partial restoration of TCTP protein expression was observed in the tumors of heterozygous TCTP mice (TCTP KD/TCTP±). The blockage of mRNA synthesis with ActD stimulated TCTP protein expression in HCC cells. In contrast, combined treatment with ActD and CHX or MG132 treatment alone did not lead to the TCTP protein accumulation in cells. Furthermore, following the introduction of exogenous TCTP in cells and orthotopic HCC tumor models, the endogenous TCTP protein did not change with the recombinational TCTP expression and kept a rather stable level. Dual-luciferase assays revealed that the coding sequence of TCTP mRNA functions as a sponge to regulate the TCTP protein expression. Collectively, our results indicated that the TCTP mRNA and protein formed a closed regulatory circuit and works as a buffering system to keep the homeostasis of TCTP protein levels in HCC.

Adoptive transfer of polarized M2c macrophages ameliorates acute rejection in rat liver transplantation.

Hepatic macrophages play pivotal roles in tolerance induction after liver transplantation (LT). However, macrophages possess functional heterogeneities, and the protective role of M2c macrophages, a macrophage subtype characterized by the surface marker CD163 that secretes interleukin-10 (IL-10) and transforming growth factor-ß1 (TGF-ß1), in acute rejection following LT, has not been addressed. The aim of this study was to determine whether polarized macrophages of the M2c subtype could improve outcomes after LT for rats, including survival rate, liver function, and inflammatory infiltration. In our study, the numbers of CD163-positive cells were found to be increased in tolerant liver grafts. Immediately following the surgery, M2c macrophages induced from rat bone marrow-derived cells were infused into recipients; this significantly improved survival rate and liver function. The expression levels of IL-10 and TGF-ß1 were markedly increased in these rats compared to those in the control group. Furthermore, CD8+ T-cell infiltration was reduced, whereas the numbers of apoptotic cells increased, in rats treated with M2c. To explore the mechanisms of the protective role of M2c, the numbers of major histocompatibility complex (MHC) class II positive cells were found to be decreased and the expression of N-acetylglucosaminyltransferase V (MGAT5) was up-regulated in M2c infusion groups. Together, these findings demonstrate that polarization of macrophages towards the M2c phenotype ameliorated acute rejection in a rat LT model and may provide a novel and effective therapeutic approach for AR after transplantation.

A 14-Year Follow-Up of a Combined Liver-Pancreas-Kidney Transplantation: Case Report and Literature Review.

Objective: To investigate the long-term effect of triple organ transplantation (liver, kidney, and pancreas) in a patient with end-stage liver disease, post chronic hepatitis B, cirrhosis, chronic renal failure, and insulin-dependent diabetes mellitus caused by chronic pancreatitis and to explore the optimal surgical procedure. Case: A 43-year-old man with progressive emaciation and hypourocrinia for 2 months. Results indicated exocrine pancreatic insufficiency and insulin-dependent diabetes related to chronic pancreatitis (CP) after developing end-stage hepatic and renal failure. Simultaneous piggyback orthotopic liver and heterotopic pancreas-duodenum and renal transplantation was performed in 2005. Pancreatic exocrine secretions were drained enterically to the jejunum, and the donor kidney was placed in the left iliac fossa. Patient was prescribed with prednisone, tacrolimus, mycophenolate mofetil, Rabbit Anti-human Thymocyte Immunoglobulin, and simulect for immunosuppression. Results: Satisfactory hepatic and pancreatic functional recovery was achieved within 7 days post-surgery. The kidney was not functional, and continuous renal replacement therapy was used. However, the donor kidney was removed at day 16 post-surgery due to acute rejection reaction. A new renal transplantation at the same position was performed, and satisfactory kidney function from the new graft was achieved 3 days later. In 14 years of follow-up, patient has not had any rejection reactions or other complications such as pancreatitis, thrombosis, and localized infections. The patient is insulin independent with normal liver and renal functions. FK506+Pred was used for immunosuppression, and the tac tough level maintained 3.0-4.5 ng/ml. Lamivudine was prescribed for long-term use to inhibit HBV virus duplication. Conclusion: Simultaneous piggyback orthotopic liver and heterotopic pancreas-duodenum and renal transplantation is a good therapeutic option for patients with exocrine pancreatic insufficiency and insulin-dependent diabetes combined with hepatic and renal failure.

Translationally controlled tumor protein exerts a proinflammatory role in acute rejection after liver transplantation.

BACKGROUND: Translationally controlled tumor protein (TCTP), which has been verified to have a proinflammatory activity, plays an important role in allergy. However, it remains unclear whether TCTP has an impact on the acute rejection (AR) after liver transplantation. METHODS: Three protocols were used to delineate the role of TCTP in AR after liver transplantation. First, in rat orthotopic liver transplantation (OLT), the expression of TCTP was measured by enzyme-linked immunosorbent assay (ELISA), real-time PCR, Western blot and immunofluorescence assays. Second, in mixed lymphocyte reaction (MLR), the role of TCTP in lymphocyte proliferation was measured by carboxyfluorescein succinimidyl ester (CFSE) labeling and the impact of TCTP on inflammatory factor release was detected by cytokine arrays. Third, in human OLT, the level of serum TCTP was detected by ELISA, and the relationship between TCTP and model for early allograft function (MEAF) score was assessed by Spearman's correlation. RESULTS: In rat OLT, AR resulted in great harm to allografts, manifesting as deterioration of liver function, increasing inflammatory factors and infiltrating lymphocytes. Meanwhile, TCTP was overexpressed in serum and allografts. Higher level of TCTP was associated with higher rejection activity index (RAI). In an MLR protocol, TCTP knockdown inhibited the proliferation of mixed inflammatory cells and significantly suppressed the release of 15 cytokines and chemokines. In human OLT, the serum TCTP was up-regulated within a week after operation. Additionally, the increasing speed of serum TCTP positively correlated with MEAF scores (r = 0.449; P = 0.0088). CONCLUSIONS: Up-regulated TCTP positively affects AR after liver transplantation.

Correction: Translationally controlled tumor protein promotes liver regeneration by activating mTORC2/AKT signaling.

Reference 26 reads "Hua, J. et al. TCTP and CSN4 control cell cycle progression and development by regulating CULLIN1 neddylation in plants and animals. PLoS Genet. 15, e1007899 (2019)." However, it should read "Betsch, L. et al. TCTP and CSN4 control cell cycle progression and development by regulating CULLIN1 neddylation in plants and animals. PLoS Genet. 15, e1007899 (2019)".

Translationally controlled tumor protein promotes liver regeneration by activating mTORC2/AKT signaling.

Translationally controlled tumor protein (TCTP), which is a protein characterized by its potent proliferation promoting activity, has been well studied in the area of growth and tumorigenesis. However, the specific role of TCTP in liver regeneration (LR) and its underlying mechanism remains unclear. In order to investigate the contribution of TCTP during LR, heterozygous TCTP mice were generated, and a mimic LR model was applied to TCTP-knockdown (KD) hepatic cell lines. The results revealed that TCTP-KD impaired LR in mice, and manifested as the following aspects: delayed proliferation of hepatocytes, accompanied by disruption of the mRNA expression of markers of the cell cycle, degenerated lipid metabolism, and abnormal immune response. Furthermore, it was found out that TCTP activated PI3K/AKT signaling by regulating mTORC2. Lastly, the increasing rate of serum TCTP positively correlated to the recovery of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) after liver resection in humans. In summary, the present study is the first to reveal the crucial role of intracellular TCTP in LR.

Immortalization of porcine hepatocytes with a α-1,3-galactosyltransferase knockout background.

BACKGROUND: In vivo pig liver xenotransplantation preclinical trials appear to have poor efficiency compared to heart or kidney xenotransplantation because of xenogeneic rejection, including coagulopathy, and particularly thrombocytopenia. In contrast, ex vivo pig liver (wild type) perfusion systems have been proven to be effective in "bridging" liver failure patients until subsequent liver allotransplantation, and transgenic (human CD55/CD59) modifications have even prolonged the duration of pig liver perfusion. Despite the fact that hepatocyte cell lines have also been proposed for extracorporeal blood circulation in conditions of acute liver failure, porcine hepatocyte cell lines, and the GalT-KO background in particular, have not been developed and applied in this field. Herein, we established immortalized wild-type and GalT-KO porcine hepatocyte cell lines, which can be used for artificial liver support systems, cell transplantation, and even in vitro studies of xenotransplantation. METHODS: Primary hepatocytes extracted from GalT-KO and wild-type pigs were transfected with SV40 LT lentivirus to establish immortalized GalT-KO porcine hepatocytes (GalT-KO-hep) and wild-type porcine hepatocytes (WT). Hepatocyte biomarkers and function-related genes were assessed by immunofluorescence, periodic acid-Schiff staining, indocyanine green (ICG) uptake, biochemical analysis, ELISA, and RT-PCR. Furthermore, the tumorigenicity of immortalized cells was detected. In addition, a complement-dependent cytotoxicity (CDC) assay was performed with GalT-KO-hep and WT cells. Cell death and viability rates were assessed by flow cytometry and CCK-8 assay. RESULTS: GalT-KO and wild-type porcine hepatocytes were successfully immortalized and maintained the characteristics of primary porcine hepatocytes, including albumin secretion, ICG uptake, urea and glycogen production, and expression of hepatocyte marker proteins and specific metabolic enzymes. GalT-KO-hep and WT cells were confirmed as having no tumorigenicity. In addition, GalT-KO-hep cells showed less apoptosis and more viability than WT cells when exposed to complement and xenogeneic serum. CONCLUSIONS: Two types of immortalized cell lines of porcine hepatocytes with GalT-KO and wild-type backgrounds were successfully established. GalT-KO-hep cells exhibited higher viability and injury resistance against a xenogeneic immune response.

Retrospective analysis of sorafenib efficacy and safety in Chinese patients with high recurrence rate of post-hepatic carcinectomy.

Background: There is no guideline recommendation for preventing hepatocellular carcinoma (HCC) recurrence after hepatic resection. Moreover, an unmet need exists on the effectiveness of sorafenib therapy in recurrent HCC. Purpose: We therefore assessed the efficacy and safety of sorafenib in Chinese HCC patients with high risk of recurrence. Patients and methods: Data were collected retrospectively from 15 Chinese research centers from January 1, 2012 to November 15, 2013, by chart reviews of patients with moderate-advanced HCC who received hepatic carcinectomy. The primary end point was recurrence-free survival rate at 1 year in patients with a high recurrence risk. Secondary end points included 1-year survival rate, time to recurrence and safety assessment. Results: A total of 209 high-risk patients (sorafenib, n=98; control, n=111) who underwent carcinectomy were analyzed. There was no significant difference in the proportion of patients with recurrence-free survival at 1 year between the sorafenib and control (70.43% vs 68.90%: χ2=0.007, P=0.934). One-year survival rate was significantly higher with sorafenib than observed with control (95.5% vs 83.35%; χ2=7.441, P=0.006). Time to recurrence between sorafenib and control groups was similar. Incidences of all the adverse events (AEs) were similar in both the groups and transaminase elevation was most common in both groups (20.37% vs 24.79%). Thrombocytopenia incidence was significantly lower with the sorafenib group than with control (1.85% vs 9.40%; P=0.015). Conclusion: Sorafenib may be considered as a feasible option in the treatment of HCC recurrence.

NOTCH Signaling via WNT Regulates the Proliferation of Alternative, CCR2-Independent Tumor-Associated Macrophages in Hepatocellular Carcinoma.

Tumor-associated macrophages (TAM) play pivotal roles in tumor progression and metastasis, but the contribution and regulation of different macrophage populations remain unclear. Here we show that Notch signaling plays distinct roles in regulating different TAM subsets in hepatocellular carcinoma (HCC). Myeloid-specific NOTCH blockade by conditional disruption of recombination signal binding protein Jκ (RBPj cKO) significantly delayed the growth of subcutaneously inoculated Lewis lung carcinoma (LLC), but accelerated orthotopically inoculated hepatic Hepa1-6 tumors in mice. In contrast to subcutaneous LLC, RBPj cKO significantly increased the number of TAMs in hepatic Hepa1-6 tumors despite impeded differentiation of monocyte-derived TAMs (moTAM). The dominating TAMs in orthotopic HCC manifested properties of Kupffer cells (KC) and hence are tentatively named KC-like TAMs (kclTAM). The increased proliferation of RBPj cKO kclTAMs was maintained even in Ccr2 -/- mice, in which moTAMs were genetically blocked. NOTCH signaling blockade accelerated proliferation of kclTAMs via enhanced ß-catenin-dependent WNT signaling, which also downregulated IL12 and upregulated IL10 expression by kclTAMs likely through c-MYC. In addition, myeloid-specific RBPj cKO facilitated hepatic metastasis of colorectal cancer but suppressed lung metastasis in mice, suggesting that the phenotype of RBPj cKO in promoting tumor growth was liver-specific. In patient-derived HCC biopsies, NOTCH signaling negatively correlated with WNT activation in CD68+ macrophages, which positively correlated with advanced HCC stages. Therefore, NOTCH blockade impedes the differentiation of moTAMs, but upregulates Wnt/ß-catenin signaling to promote the proliferation and protumor cytokine production of kclTAMs, facilitating HCC progression and hepatic metastasis of colorectal cancer. SIGNIFICANCE: These findings highlight the role of NOTCH and WNT signaling in regulating TAMs in hepatocellular carcinoma.

Downregulation of CENPK suppresses hepatocellular carcinoma malignant progression through regulating YAP1.

BACKGROUND: Several studies have found that centromere protein K (CENPK) is overexpressed in several tumour types and promotes tumor progression. However, there has been little research on the role of CENPK in the progression of hepatocellular carcinoma (HCC). MATERIALS AND METHODS: The expression of CENPK in HCC tissues was quantified by Western blot and quantitative real-time PCR. Cells were transfected with lentiviral plasmids containing shRNA sequences targeting CENPK and YAP1 to silence the expression of CENPK and YAP1. Cell Counting Kit-8 assay, colony formation assay, wound healing assay, and transwell invasion assay were performed to evaluate cell growth, migration, and invasion of HCC cells. Tumorigenicity assay was used to detect the effect of CENPK on the growth of HCC cells. Western blot assay was performed to investigate the expression of epithelial-mesenchymal transition (EMT) markers and YAP1. RESULTS: Compared to that in adjacent non-tumor tissues, CENPK was aberrantly upregulated in HCC tumor tissues. Furthermore, CENPK knockdown significantly inhibited proliferation, migration, invasion, and EMT progression in HCC cells. Mechanistically, we identified that YAP1 was responsible for the tumor-suppressive effects of CENPK knockdown in the HCC cells. The inhibitory effects of CENPK silencing on cell proliferation, migration, invasion, and EMT were partially reversed by the restoration of YAP1 expression. CONCLUSION: Our results suggested that the CENPK-YAP1-EMT axis plays a critical role in regulating HCC malignant progression, indicating the role of this axis as a potential therapeutic target for HCC.

ERRATUM.

MicroRNA-200a (miR-200a) is frequently downregulated in most cancer types and plays an important role in carcinogenesis and cancer progression. In this study, we determined that miR-200a was downregulated in hepatocellular carcinoma (HCC) tissues and cell lines, consistent with the results of our previous study. Because a previous study suggested that downregulation of miR-200a is correlated with HCC metastasis, we aimed to elucidate the mechanism underlying the role of miR-200a in metastasis in HCC. Here we observed that overexpression of miR-200a resulted in suppression of HCC metastatic ability, including HCC cell migration, invasion, and metastasis, in vitro and in vivo. Furthermore, bioinformatics and luciferase reporter assays indicated that GAB1 is a direct target of miR-200a. Inhibition of GAB1 resulted in substantially decreased cell invasion and migration similar to that observed with overexpression of miR-200a in HCC cell lines, whereas restoration of GAB1 partially rescued the inhibitory effects of miR-200a. Taken together, these data provide novel information for comprehending the tumor-suppressive role of miR-200a in HCC pathogenesis through inhibition of GAB1 translation.

Berberine Inhibits Growth of Liver Cancer Cells by Suppressing Glutamine Uptake.

INTRODUCTION: Glutamine metabolism is essential for the proliferation of cancer cells. Transported by SLC1A5, a Na+ dependent transporter, glutamine is absorbed for further use. Recent studies have revealed the anti-tumor effect of berberine. The present study aimed to evaluate the effect of berberine on cancer cell glutamine metabolism. MATERIALS AND METHODS: The inhibitory effect of berberine on liver cancer cells was analyzed by CCK-8 and EdU assay. The glutamine concentrations were detected by ELISA and UHPLC-MRM-MS analysis. Glutamine metabolism-related proteins were determined by Western blot, immunofluorescent analysis and immunohistochemistry. RESULTS: Berberine inhibited the proliferation of Hep3B and BEL-7404 cell in vitro. Berberine suppressed the glutamine uptake by inhibiting SLC1A5. The upregulation of SLC1A5 led to an increased glutamine uptake and improved tolerance to berberine. Berberine suppresses SLC1A5 expression by inhibiting c-Myc. Furthermore, berberine suppresses the growth of tumor xenografts, and the expression of SLC1A5 and c-Myc in vivo. The high expression of SLC1A5 in hepatocellular carcinoma (HCC) tissues is associated with poor prognosis. CONCLUSION: Berberine can suppress the proliferation of liver cancer cells by reducing SLC1A5 expression.